Methods for modulating telomerase activity

ABSTRACT

The present invention relates to isolated or purified molecule(s) capable of binding to one or more of telomeric, G-quadruplex, or G-quartet nucleic acid(s).

FIELD OF THE INVENTION

[0001] The invention relates to DNA binding molecules. In particular theinvention relates to molecules which bind to G-quadruplex or telomericDNA.

BACKGROUND TO THE INVENTION

[0002] There is considerable interest in molecules that bind totelomeric DNA sequences and G-quadruplexes. Such molecules will beuseful to test hypotheses of telomere length regulation, and may havetherapeutic potential.

[0003] Several naturally occurring proteins with affinity forG-quadruplexes have been described in the prior art (reviewed inWellinger. R. J., & Sen, D. (1997) European Journal of Cancer 33,735-749), although none have so far proved to be good candidates for useas diagnostic probes or therapeutic tools.

[0004] Prior art quadruplex DNA binding molecules, such as a recentlyreported DNA-binding autoantibody (Brown, B. A., Li, Y. Q., Brown, J.C., Hardin, C. C., Roberts, J. F., Pelsue, S. C., & Shultz., L. D.(1998) Biochemistry 37, 16325-16337), have only moderate bindingaffinities and discriminate weakly between duplex and quadruplex DNA.

[0005] DNA binding molecules are disclosed in M. D. Isalan, A. Klug andY. Choo, International Patent Application Publication No. WO98/53057.

[0006] Naturally occurring telomere-binding proteins are also unable todiscriminate these structures. For example, Saccharomyces cerevisiaeRAP1 (Giraldo, R. & Rhodes, D. (1994) EMBO J 13, 2411-2420) has distinctbut inseparable domains for binding quadruplexes and double strandedDNA.

[0007] The present invention seeks to overcome problems associated withthe prior art.

SUMMARY OF THE INVENTION

[0008] Disclosed herein is the engineering of DNA-binding polypeptidemolecule(s) that bind to telomeric G-quadruplex structure(s).Preferably, these molecules are polypeptides comprising a zinc fingermotif.

[0009] Zinc finger polypeptides according to the present inventionadvantageously bind to single stranded human telomeric DNA with anaffinity comparable to the binding of naturally occurring transcriptionfactors to their cognate duplex DNA recognition site(s). DNA in thebound complexes is preferably in the G-quadruplex conformation.

[0010] Thus, in a first aspect, the invention relates to an isolated orpurified molecule capable of binding to one or more of telomeric,G-quadruplex, or G-quartet nucleic acid.

[0011] As used herein, the term ‘isolated or purified’ is used to meanthat a molecule is free of one or more components of its naturalenvironment. Where the molecule(s) are produced in vitro or in vivo in alaboratory, they are considered to be isolated or purified. Isolatedmolecules according to the invention therefore include such moleculeswhen produced using recombinant cell culture, phage culture etc.Molecules present in an organism expressing a recombinant nucleic acidencoding same, whether the molecule(s) are “isolated” or otherwise, arealso included within the scope of the present invention.

[0012] The term ‘molecule’ has its natural meaning. Preferably,molecules according to the invention are polypeptides.

[0013] The expression ‘capable of binding to one or more of’ is used toindicate that the molecule(s) retain the ability to associate with,interact with, or bind to one or more of the mentioned entities. Thisbinding may be reversible or irreversible. This binding may be temporaryor permanent. It may be covalent, ionic, or hydrogen bonding,van-der-waals association or any other type of molecular interaction.

[0014] Telomeric nucleic acid refers to nucleic acid comprised in orderived from telomeres of eukaryotic cells. The term therefore includesknown telomeric repetitive DNA sequences (see below for examples), mayinclude related RNA sequences such as telomeric primer sequences, andmay include sub-telomeric repeat sequences, or other sequence(s) foundat chromosome ends. The term is intended to include these nucleic acidsregardless of their molecular context. This means that such moleculesare included if they are in a complex with telomeric or scaffoldproteins, or if they are naked in vitro. The molecules are included whenthey are in vivo such as bona fide telomeres in cell nuclei, or whenthey are removed from their natural context, such as when on a chef gelor when cloned. The term telomeric nucleic acid may also includemutants, fragments or derivatives thereof, provided such mutants,fragments or derivatives retain substantial sequence homology with saidtelomeric nucleic acid molecules—this is discussed in more detail below.

[0015] Telomeric nucleic acids are known to adopt unconventional ornon-conventional structural conformations, mediated by unusualbase-pairing (ie. other than simple base paired duplex DNA). Examples ofthese structures include G-quadruplexes.

[0016] The term ‘G-quadruplex’ as understood herein relates to anyfour-stranded DNA structure. Those skilled in the art realise that thesestructures comprise loops and hairpins and such like as the two strandsof a duplex fold back alongside themselves to form a four-strandedstructure, even though only two distinct nucleotide polymer strands maybe present. It is also understood that such structures may comprisesingle-stranded DNA and/or double stranded DNA. Accordingly, in anotheraspect, the invention relates to a nucleic acid binding molecule asdescribed above wherein said nucleic acid comprises single-stranded DNA.The feature which characterises a ‘G-quadruplex’ as the term is usedherein is that at least a part of the structure to which it refers is ina four-stranded conformation. G-quadruplexes may be intra- orintermolecular.

[0017] The term ‘G-quartet’ refers to that part of a nucleic acidstructure which is in a four-stranded conformation. A G-quartet istherefore any segment of nucleic acid or combination of nucleic acidswhich is in a four-stranded conformation.

[0018] Thus, in another aspect, the invention relates to a nucleic acidbinding molecule as described above wherein said nucleic acid is not ina double-helical conformation.

[0019] Four-stranded nucleic acid conformations (ie. G-quartets) maycomprise unconventional base pairing. Conventional base pairing isconsidered to be Watson and Crick double helical base paired nucleicacid. Unconventional base pairing is therefore base pairing other thanWatson and Crick double helical base pairing. Thus, in another aspect,the invention relates to a nucleic acid binding molecule as describedabove wherein said nucleic acid is in a non-Watson-Crick base pairedconformation.

[0020] An example of unconventional base pairing is Hoogsteen basepairing. Thus, in another aspect, the invention relates to a nucleicacid binding molecule as described above wherein said nucleic acidcomprises Hoogsteen base pairing.

[0021] In another aspect, the invention relates to a nucleic acidbinding molecule as described above wherein said nucleic acid iscomprised in a chromosome end.

[0022] In another aspect, the invention relates to a nucleic acidbinding molecule as described above wherein said nucleic acid iscomprised in a telomeric structure.

[0023] Preferably, molecules according to the invention arepolypeptides. Thus, in another aspect, the invention relates to anucleic acid binding molecule as described above wherein said moleculeis a polypeptide.

[0024] More preferably molecules according to the invention arepolypeptides comprising a zinc finger motif. A zinc finger is aDNA-binding protein domain that may be used as a scaffold to designDNA-binding proteins. Preferably, the molecule of the invention is apolypeptide comprising a zinc finger nucleic acid binding motif. Theproperties of such motifs include the possession of a Cys2-His2 motif,and are discussed in more detail below. Therefore, in another aspect,the invention relates to a nucleic acid binding molecule as describedabove wherein said molecule is a polypeptide comprising at least onezinc finger motif.

[0025] Molecules of the present invention preferably exhibit strongdiscrimination between G-quadruplex nucleic acid and the double-strandedform of the same sequence and between G-quadruplex nucleic acid and thesingle-stranded variants.

[0026] Accordingly, in another aspect, the invention relates to anucleic acid binding molecule as described above wherein said moleculehas an affinity for G-quadruplex nucleic acid which is different fromits affinity for duplex nucleic acid. Preferably said molecule has anaffinity for G-quadruplex nucleic acid which is higher than its affinityfor duplex nucleic acid.

[0027] In another aspect, the invention relates to a method for assayingtelomerase activity, said method comprising providing a sample ofnucleic acid substrate for telomerase; contacting said sample with amolecule as described above; monitoring the binding of said molecule tosaid nucleic acid sample; contacting said nucleic acid sample with atelomerase; contacting said nucleic acid sample with a molecule asdescribed above; monitoring the binding of said molecule to saidtelomerase treated nucleic acid sample, and comparing the binding of thefirst monitoring step with the binding of the second monitoring step.

[0028] This or other aspect(s) may comprise dispensing a nucleic acidsample into the wells of a plate suitable for use with an ELISA reader,such as a 96-well microtitre plate. Gq1* labelled with fluorescent dyeor enzyme is then added to the well, incubated and washed, and thebinding of the Gq1* molecules to the nucleic acid sample is measured byfluorescence or ELISA. The telomerase or candidate telomerase is addedto the nucleic acid sample, and incubated at a suitable temperature forthe telomerase or candidate telomerase to function. Fresh Gq1* labelledwith fluorescent dye or enzyme is then added to the well, incubated andwashed, and the binding of the Gq1* molecules to the nucleic acid sampleis measured by fluorescence or ELISA. The binding of the Gq1* moleculesaccording to the invention to the nucleic acid sample before and aftertreatment with the telomerase or candidate telomerase is compared. Ahigher binding coefficient after telomerase treatment indicates thatmore target nucleic acid is present after telomerase treatment, and thusindicates that telomerase activity was indeed present in the sample.This method can be easily adapted for estimating the length oftelomere(s), by simply measuring the binding of an excess of moleculesaccording to the invention to normalised masses of nucleic acid sample.The amount of bound molecule per given mass of DNA then provides anestimate of the length of the telomere(s), if any are present.

[0029] Thus, in another aspect, the invention relates to a method forestimating the length of telomere(s), said method comprising contactingsaid telomere(s) with a molecule as described above; monitoring thebinding of said molecule to said telomere sample, and estimating thelength of said telomeres from the strength of said binding.

[0030] In another aspect, the invention relates to a method for assayingtelomerase activity, said method comprising; providing a sample ofnucleic acid substrate for telomerase; contacting said nucleic acidsample with a telomerase; contacting said nucleic acid sample with amolecule as described above, and monitoring the binding of said moleculeto said telomerase treated nucleic acid sample. This is discussed inmore detail below, such as in the Examples section.

[0031] In another aspect, the invention relates to a method fordiscriminating between duplex and quadruplex nucleic acid comprisingcontacting a sample of nucleic acid with a molecule as described above,and monitoring the binding of said molecule to said nucleic acid.‘Discriminating between’ means that the two or more entities which arebeing discriminated may be told apart or mutually excluded or identifiedor otherwise distinguished. In this example, the term is used to meanthat duplex nucleic acid and quadruplex nucleic acid may bedistinguished using this method.

[0032] In another aspect, the invention relates to a method fordetecting telomeric structures in vivo comprising contacting a labelledmolecule as described above with a sample, and monitoring said labelledmolecule. The molecule may be labelled using any suitable method as arewell known in the art and include fluorescent labelling, radioactivelabelling, peptide tagging, immunolabelling and the like. These arediscussed in more detail below,

[0033] In another aspect, the invention relates to a method formanipulating telomeric structure(s) in vivo comprising contacting alabelled molecule as described above with a telomeric structure, whereinsaid molecule further comprises an effector domain. In this context,‘manipulating’ means altering, binding, cleaving, modifying (such aschemical and/or enzymatic modification) or similar effect. An effectordomain may be a repressor domain, a nuclease, a tag, an enzyme orenzymatic activity, a toxin, a prodrug or any other suitable effector asdiscussed below.

[0034] In another embodiment, the present invention relates to thedesign and selection of zinc fingers that bind single stranded telomericDNA in the G-quadruplex conformation.

[0035] In another aspect, the invention relates to a method as describedabove wherein said assay method comprises an ELISA assay.

[0036] In another aspect, the invention relates to a method as describedabove wherein said assay method is in micro-well format.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The term “library” is used according to its common usage in theart, to denote a collection of polypeptides or, preferably, nucleicacids encoding polypeptides. The polypeptides of the invention containregions of randomisation, such that each library will comprise or encodea repertoire of polypeptides, wherein individual polypeptides differ insequence from each other. The same principle is present in virtually alllibraries developed for selection, such as by phage display.

[0038] Randomisation, as used herein, refers to the variation of thesequence of the polypeptides which comprise the library, such thatvarious amino acids may be present at any given position in differentpolypeptides. Randomisation may be complete, such that any amino acidmay be present at a given position, or partial, such that only certainamino acids are present. Preferably, the randomisation is achieved bymutagenesis at the nucleic acid level, for example by synthesising novelgenes encoding mutant proteins and expressing these to obtain a varietyof different proteins. Alternatively, existing genes can be themselvesmutated, such by site-directed or random mutagenesis, in order to obtainthe desired mutant genes

Variants

[0039] The nucleic acid binding polypeptide molecule as provided by thepresent invention includes splice variants encoded by mRNA generated byalternative splicing of a primary transcript, amino acid mutants,glycosylation variants and other covalent derivatives of said moleculewhich retain the physiological and/or physical properties of saidmolecule, such as its nucleic acid binding activity. Exemplaryderivatives include molecules wherein the protein of the invention iscovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid. Such a moiety may be a detectable moiety such as an enzyme or aradioisotope, or may be a molecule capable of facilitating crossing ofcell membrane(s) etc.

[0040] Derivatives can be fragments of the nucleic acid bindingmolecule. Fragments of said molecule comprise individual domainsthereof, as well as smaller polypeptides derived from the domains.Preferably, smaller polypeptides derived from the molecule according tothe invention define a single epitope which is characteristic of saidmolecule. Fragments may in theory be almost any size, as long as theyretain one characteristic of the nucleic acid binding molecule.Preferably, fragments may be at least 3 amino acids and in length.

[0041] Derivatives of the nucleic acid binding molecule also comprisemutants thereof, which may contain amino acid deletions, additions orsubstitutions, subject to the requirement to maintain at least onefeature characteristic of said molecule. Thus, conservative amino acidsubstitutions may be made substantially without altering the nature ofthe molecule, as may truncations from the N- or C-terminal ends, or thecorresponding 5′- or 3′-ends of a nucleic acid encoding it. Deletions orsubstitutions may moreover be made to the fragments of the moleculecomprised by the invention. Nucleic acid binding molecule mutants may beproduced from a DNA encoding a transcription protein which has beensubjected to in vitro mutagenesis resulting e.g. in an addition,exchange and/or deletion of one or more amino acids. For example,substitutional, deletional or insertional variants of the molecule canbe prepared by recombinant methods and screened for nucleic acid bindingactivity as described herein.

[0042] The fragments, mutants and other derivatives of the polypeptidenucleic acid binding molecule preferably retain substantial homologywith said molecule. As used herein. “homology” means that the twoentities share sufficient characteristics for the skilled person todetermine that they are similar in origin and/or function. Preferably,homology is used to refer to sequence identity. Thus, the derivatives ofthe molecule preferably retain substantial sequence identity with thesequence of said molecule.

[0043] “Substantial homology”, where homology indicates sequenceidentity, means more than 75% sequence identity and most preferably asequence identity of 90% or more.

[0044] Amino acid sequence identity may be assessed by any suitablemeans, including the BLAST comparison technique which is well known inthe art, and is described in Ausubel et al., Short Protocols inMolecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc.

Mutations

[0045] Mutations may be performed by any method known to those of skillin the art. Preferred, however, is site-directed mutagenesis of anucleic acid sequence encoding the protein of interest. A number ofmethods for site-directed mutagenesis are known in the art, from methodsemploying single-stranded phage such as M13 to PCR-based techniques (see“PCR Protocols: A guide to methods and applications”, M. A. Innis, D. H.Gelfand, J. J. Sninsky, T. J. White (eds.), Academic Press, New York,1990), Preferably, the commercially available Altered Site IIMutagenesis System (Promega) may be employed, according to thedirections given by the manufacturer.

[0046] Screening of the proteins produced by mutant genes is preferablyperformed by expressing the genes and assaying the binding ability ofthe protein product. A simple and advantageously rapid method by whichthis may be accomplished is by phage display, in which the mutantpolypeptides are expressed as fusion proteins with the coat proteins offilamentous bacteriophage, such as the minor coat protein pH ofbacteriophage m13 or gene III of bacteriophage Fd, and displayed on thecapsid of bacteriophage transformed with the mutant genes. The targetnucleic acid sequence is used as a probe to bind directly to the proteinon the phage surface and select the phage possessing advantageousmutants, by affinity purification. The phage are then amplified bypassage through a bacterial host, and subjected to further rounds ofselection and amplification in order to enrich the mutant pool for thedesired phage and eventually isolate the preferred clone(s). Detailedmethodology for phage display is known in the art and set forth, forexample, in U.S. Pat. No. 5,223,409; Choo and Klug, (1995) CurrentOpinions in Biotechnology 6:431-436; Smith, (1985) Science228:1315-1317; and McCafferty et al., (1990) Nature 348:552-554; allincorporated herein by reference. Vector systems and kits for phagedisplay are available commercially, for example from Pharmacia.

[0047] The present invention allows the production of what areessentially artificial nucleic acid binding proteins. In these proteins,artificial analogues of amino acids may be used, to impart the proteinswith desired properties or for other reasons. Thus, the term “aminoacid”, particularly in the context where “any amino acid” is referredto, means any sort of natural or artificial amino acid or amino acidanalogue that may be employed in protein construction according tomethods known in the art. Moreover, any specific amino acid referred toherein may be replaced by a functional analogue thereof, particularly anartificial functional analogue. The nomenclature used herein thereforespecifically comprises within its scope functional analogues of thedefined amino acids.

[0048] Molecules according to the invention are preferably zinc fingerpolypeptides. In other words, they comprise a Cys2-His2 zinc fingermotif.

Zinc Fingers

[0049] A zinc finger is a DNA-binding protein domain that may be used asa scaffold to design DNA-binding proteins with predeterminedsequence-specificity (Klug, A. & Rhodes, D. (1987) ‘Zinc fingers’: anovel protein motif for nucleic acid recognition. Trends Biochem. Sci.12, 464-469; Choo, Y. & Klug, A. (1995) Designing DNA-binding proteinson the surface of filamentous phage. Curr. Opin. Biotech. 6, 431-436).The peptide motif comprises about 30 amino acids that adopt a compactDNA-binding structure on chelating a zinc ion (Miller, J., McLachlan, A.D. & Klug, A. (1985) Repetitive zinc-binding domains in the proteintranscription factor IIIA from Xenopus oocytes. EMBO J 4, 1609-1614).Each zinc finger module is capable of recognising 3-4 bp of DNA, suchthat arrays comprising tandemly repeated modules bind proportionallylonger nucleotide sequences. The crystal structure of the Zif268DNA-binding domain, in complex with its optimal DNA binding site, showsthat the zinc finger array wraps around the DNA, with the α-helix ofeach finger buried in the major groove (Pavletich, N. P. & Pabo, C. O.(1991) Zinc finger-DNA recognition: Crystal structure of a Zif268-DNAcomplex at 2.1 Å. Science 252, 809-817).

[0050] The geometrical properties of zinc finger structures mean that aversatile binding surface can be created by varying a small number ofamino acid positions on each finger's central α-helix. Moreover, zincfingers may be linked together to bind to longer, contiguous stretchesof DNA. Large randomised libraries of zinc fingers have been engineeredby phage display, so that zinc finger variants are displayed on theviral capsid. Such libraries have been extensively screened to selectfingers that bind to various duplex DNA sequences (Choo, Y., & Klug, A.(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11163-11167. Greisman, H. A., &Pabo, C. O. (1997) Science 275, 657-661. Jamieson, A. C., Kim, S. -H., &Wells, J. A. (1994) Biochemistry 33, 5689-5695. Wu, H., Yang, W. -P., &Barbas III, C. F. (1995) Proc. Natl. Acad. Sci. USA 92, 344-348. Isalan,M., Klug, A., & Choo, Y. (1998) Biochemistry 37, 12026-12033.) and toRNA (Friesen, W. J., & Darby, M. K. (1997) J Biol Chem 272, 10994-10997.Friesen, W. J., & Darby, M. K. (1998) Nat Struct Biol 5, 543-546.Blancafort, P., Steinberg, S. V., Paquin, B., Klinck, R., Scott, J. K.,& Cedergren, R. (1999) Chemistry and Biology 6, 585-597.).

[0051] Zinc fingers, as is known in the art, are nucleic acid bindingmolecules. A zinc finger binding motif is a structure well known tothose in the art and defined in, for example, Miller et al., (1985) EMBOJ. 4:1609-1614; Berg (1988) PNAS (USA) 85:99-102; Lee et al., (1989)Science 245:635-637; see International patent applications WO 96/06166and WO 96/32475, corresponding to U.S. Ser. No. 08/422,107, incorporatedherein by reference.

[0052] As used herein. “nucleic acid” refers to both RNA and DNA,constructed from natural nucleic acid bases or synthetic bases, ormixtures thereof. Preferably, however, the binding proteins of theinvention are DNA binding proteins.

[0053] All of the nucleic acid-binding residue positions of zincfingers, as referred to herein, are numbered from the first residue inthe α-helix of the finger, ranging from +1 to +9. “−1” refers to theresidue in the framework structure immediately preceding the α-helix ina, Cys2-His2 zinc finger polypeptide. Cys2-His2 zinc finger bindingproteins, as is well known in the art, bind to target nucleic acidsequences via α-helical zinc metal atom co-ordinated binding motifsknown as zinc fingers.

[0054] These and other considerations may be incorporated in a libraryset in accordance with the invention.

Vectors

[0055] The nucleic acid encoding the nucleic acid binding proteinaccording to the invention can be incorporated into vectors for furthermanipulation. As used herein, vector (or plasmid) refers to discreteelements that are used to introduce heterologous nucleic acid into cellsfor either expression or replication thereof. Selection and use of suchvehicles are well within the skill of the person of ordinary skill inthe art. Many vectors are available, and selection of appropriate vectorwill depend on the intended use of the vector, i.e. whether it is to beused for DNA amplification or for nucleic acid expression, the size ofthe DNA to be inserted into the vector, and the host cell to betransformed with the vector. Each vector contains various componentsdepending on its function (amplification of DNA or expression of DNA)and the host cell for which it is compatible. The vector componentsgenerally include, but are not limited to, one or more of the following:an origin of replication, one or more marker genes, an enhancer element,a promoter, a transcription termination sequence and a signal sequence.

[0056] Both expression and cloning vectors generally contain nucleicacid sequence that enable the vector to replicate in one or moreselected host cells. Typically in cloning vectors, this sequence is onethat enables the vector to replicate independently of the hostchromosomal DNA, and includes origins of replication or autonomouslyreplicating sequences. Such sequences are well known for a variety ofbacteria, yeast and viruses. The origin of replication from the plasmidpBR322 is suitable for most Gram-negative bacteria, the 2μ plasmidorigin is suitable for yeast, and various viral origins (e.g. SV 40,polyoma, adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication component is not needed formammalian expression vectors unless these are used in mammalian cellscompetent for high level DNA replication, such as COS cells.

[0057] Most expression vectors are shuttle vectors, i.e. they arecapable of replication in at least one class of organisms but can betransfected into another class of organisms for expression. For example,a vector is cloned in E. coli and then the same vector is transfectedinto yeast or mammalian cells even though it is not capable ofreplicating independently of the host cell chromosome. DNA may also bereplicated by insertion into the host genome. However, the recovery ofgenomic DNA encoding the nucleic acid binding protein is more complexthan that of exogenously replicated vector because restriction enzymedigestion is required to excise nucleic acid binding protein DNA. DNAcan be amplified by PCR and be directly transfected into the host cellswithout any replication component.

Selectable Markers

[0058] Advantageously, an expression and cloning vector may contain aselection gene also referred to as selectable marker. This gene encodesa protein necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

[0059] As to a selective gene marker appropriate for yeast, any markergene can be used which facilitates the selection for transformants dueto the phenotypic expression of the marker gene. Suitable markers foryeast are, for example, those conferring resistance to antibiotics G418,hygromycin or bleomycin, or provide for prototrophy in an auxotrophicyeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.

[0060] Since the replication of vectors is conveniently done in E. coli,an E. coli genetic marker and an E. coli origin of replication areadvantageously included. These can be obtained from E. coli plasmids,such as pBR322, Bluescript© vector or a pUC plasmid, e.g. pUC18 orpUC19, which contain both E. coli replication origin and E. coli geneticmarker conferring resistance to antibiotics, such as ampicillin.

[0061] Suitable selectable markers for mammalian cells are those thatenable the identification of cells competent to take up nucleic acidbinding protein nucleic acid, such as dihydrofolate reductase (DHFR,methotrexate resistance), thymidine kinase, or genes conferringresistance to G418 or hygromycin. The mammalian cell transformants areplaced under selection pressure which only those transformants whichhave taken up and are expressing the marker are uniquely adapted tosurvive. In the case of a DHFR or glutamine synthase (GS) marker,selection pressure can be imposed by culturing the transformants underconditions in which the pressure is progressively increased, therebyleading to amplification (at its chromosomal integration site) of boththe selection gene and the linked DNA that encodes the nucleic acidbinding protein. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth, togetherwith closely associated genes which may encode a desired protein, arereiterated in tandem within the chromosomes of recombinant cells.Increased quantities of desired protein are usually synthesised fromthus amplified DNA.

Expression

[0062] Expression and cloning vectors usually contain a promoter that isrecognised by the host organism and is operably linked to nucleic acidbinding protein encoding nucleic acid. Such a promoter may be inducibleor constitutive. The promoters are operably linked to DNA encoding thenucleic acid binding protein by removing the promoter from the sourceDNA by restriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native nucleic acid binding proteinpromoter sequence and many heterologous promoters may be used to directamplification and/or expression of nucleic acid binding protein encodingDNA.

[0063] Promoters suitable for use with prokaryotic hosts include, forexample, the β-lactamase and lactose promoter systems, alkalinephosphatase, the tryptophan (Trp) promoter system and hybrid promoterssuch as the tac promoter. Their nucleotide sequences have beenpublished, thereby enabling the skilled worker operably to ligate themto DNA encoding nucleic acid binding protein, using linkers or adaptersto supply any required restriction sites. Promoters for use in bacterialsystems will also generally contain a Shine-Delgarno sequence operablylinked to the DNA encoding the nucleic acid binding protein.

[0064] Preferred expression vectors are bacterial expression vectorswhich comprise a promoter of a bacteriophage such as phagex or T7 whichis capable of functioning in the bacteria. In one of the most widelyused expression systems, the nucleic acid encoding the fusion proteinmay be transcribed from the vector by T7 RNA polymerase (Studier et al,Methods in Enzymol. 185; 60-89, 1990). In the E. coli BL21(DE3) hoststrain, used in conjunction with pET vectors, the T7 RNA polymerase isproduced from the λ-lysogen DE3 in the host bacterium, and itsexpression is under the control of the IPTG inducible lac UV5 promoter.This system has been employed successfully for over-production of manyproteins. Alternatively the polymerase gene may be introduced on alambda phage by infection with an int-phage such as the CE6 phage whichis commercially available (Novagen, Madison, USA). other vectors includevectors containing the lambda PL promoter such as PLEX (Invitrogen, NL),vectors containing the trc promoters such as pTrcHisXpress™ (Invitrogen)or pTrc99 (Pharmacia Biotech. SE) or vectors containing the tac promotersuch as pKK223-3 (Pharmacia Biotech) or PMAL (New England Biolabs,Mass., USA).

[0065] Moreover, the nucleic acid binding protein molecule according tothe invention preferably includes a secretion sequence in order tofacilitate secretion of the polypeptide from bacterial hosts, such thatit will be produced as a soluble native peptide rather than in aninclusion body. The peptide may be recovered from the bacterialperiplasmic space, or the culture medium, as appropriate. A “leader”peptide may be added to the N-terminal finger. Preferably, the leaderpeptide is MAEEKP.

[0066] Suitable promoting sequences for use with yeast hosts may beregulated or constitutive and are preferably derived from a highlyexpressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus,the promoter of the TRP1 gene, the ADHI or ADHH gene, the acidphosphatase (PH05) gene, a promoter of the yeast mating pheromone genescoding for the a- or α-factor or a promoter derived from a gene encodinga glycolytic enzyme such as the promoter of the enolase,glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glyceratekinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triose phosphate isomerase, phosphoglucose isomerase orglucokinase genes, or a promoter from the TATA binding protein (TBP)gene can be used. Furthermore, it is possible to use hybrid promoterscomprising upstream activation sequences (UAS) of one yeast gene anddownstream promoter elements including a functional TATA box of anotheryeast gene, for example a hybrid promoter including the UAS(s) of theyeast PH05 gene and downstream promoter elements including a functionalTATA box of the yeast GAP gene (PH05-GAP hybrid promoter). A suitableconstitutive PHO5 promoter is e.g. a shortened acid phosphatase PH05promoter devoid of the upstream regulatory elements (UAS) such as thePH05 (−173) promoter element starting at nucleotide −173 and ending atnucleotide −9 of the PH05 gene.

[0067] Nucleic acid binding protein gene transcription from vectors inmammalian hosts may be controlled by promoters derived from the genomesof viruses such as polyoma virus, adenovirus, fowlpox virus, bovinepapilloma virus, avian sarcoma virus, cytomegalovirus (CMV), aretrovirus and Simian Virus 40 (SV40), from heterologous mammalianpromoters such as the actin promoter or a very strong promoter, e.g. aribosomal protein promoter, and from the promoter normally associatedwith nucleic acid binding protein sequence, provided such promoters arecompatible with the host cell systems.

[0068] Transcription of a DNA encoding nucleic acid binding protein byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are relatively orientation and positionindependent. Many enhancer sequences are known from mammalian genes(e.g. elastase and globin). However, typically one will employ anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270) and theCMV early promoter enhancer. The enhancer may be spliced into the vectorat a position 5′ or 3′ to nucleic acid binding protein DNA, but ispreferably located at a site 5′ from the promoter.

[0069] Advantageously, a eukaryotic expression vector encoding a nucleicacid binding protein according to the invention may comprise a locuscontrol region (LCR). LCRs are capable of directing high-levelintegration site independent expression of transgenes integrated intohost cell chromatin, which is of importance especially where the nucleicacid binding protein gene is to be expressed in the context of apermanently-transfected eukaryotic cell line in which chromosomalintegration of the vector has occurred, or in transgenic animals.

[0070] Eukaryotic vectors may also contain sequences necessary for thetermination of transcription and for stabilising the mRNA. Suchsequences are commonly available from the 5′ and 3′ untranslated regionsof eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslaatedportion of the mRNA encoding nucleic acid binding protein.

[0071] An expression vector includes any vector capable of expressingnucleic acid binding protein nucleic acids that are operatively linkedwith regulatory sequences, such as promoter regions, that are capable ofexpression of such DNAs. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector, that upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those with ordinaryskill in the art and include those that are replicable in eukaryoticand/or prokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. For example, DNAs encoding nucleicacid binding protein may be inserted into a vector suitable forexpression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vectorsuch as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

[0072] Particularly useful for practising the present invention areexpression vectors that provide for the transient expression of DNAencoding nucleic acid binding protein in mammalian cells. Transientexpression usually involves the use of an expression vector that is ableto replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector, and, in turn,synthesises high levels of nucleic acid binding protein. For thepurposes of the present invention, transient expression systems areuseful e.g. for identifying nucleic acid binding protein mutants, toidentity potential phosphorylation sites, or to characterise functionaldomains of the protein.

[0073] Construction of vectors according to the invention employsconventional ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required. If desired, analysis to confirm correct sequences inthe constructed plasmids is performed in a known fashion.

[0074] Suitable methods for constructing expression vectors, preparingin vitro transcripts, introducing DNA into host cells, and performinganalyses for assessing nucleic acid binding protein expression andfunction are known to those skilled in the art. Gene presence,amplification and/or expression may be measured in a sample directly,for example, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA, dot blotting (DNA or RNAanalysis), or in situ hybridisation, using an appropriately labelledprobe which may be based on a sequence provided herein. Those skilled inthe art will readily envisage how these methods may be modified, ifdesired.

[0075] In accordance with another embodiment of the present invention,there are provided cells containing the above-described nucleic acids.Such host cells such as prokaryote, yeast an(i higher eukaryote cellsmay be used for replicating DNA and producing the nucleic acid bindingprotein. Suitable prokaryotes include eubacteria, such as Gram-negativeor Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains.DLH5a and HB101, or Bacilli. Further hosts suitable for the nucleic acidbinding protein encoding vectors include eukaryotic microbes such asfilamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Highereukaryotic cells include insect and vertebrate cells, particularlymammalian cells including human cells or nucleated cells from othermulticellular organisms. In recent years propagation of vertebrate cellsin culture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are epithelial or fibroblastic celllines such as Chinese hamster ovary (CHO) cells NIH 3T3 cells. HeLacells or 293T cells. The host cells referred to in this disclosurecomprise cells in in vitro culture as well as cells that are within ahost animal.

[0076] DNA may be stably incorporated into cells or may be transientlyexpressed using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene, and growing the transfected cells underconditions selective for cells expressing the marker gene. To preparetransient transfectants, mammalian cells are transfected with a reportergene to monitor transfection efficiency.

[0077] To produce such stably or transiently transfected cells, thecells should be transfected with a sufficient amount of the nucleic acidbinding protein-encoding nucleic acid to form the nucleic acid bindingprotein. The precise amounts of DNA encoding the nucleic acid bindingprotein may be empirically determined and optimised for a particularcell and assay.

[0078] Host cells are transfected or, preferably, transformed with theabove-captioned expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Heterologous DNA may be introduced intohost cells by any method known in the art, such as transfection with avector encoding a heterologous DNA by the calcium phosphatecoprecipitation technique or by electroporation. Numerous methods oftransfection are known to the skilled worker in the field. Successfultransfection is generally recognised when any indication of theoperation of this vector occurs in the host cell. Transformation isachieved using standard techniques appropriate to the particular hostcells used.

[0079] Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g. Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).

[0080] Transfected or transformed cells are cultured using media andculturing methods known in the art, preferably under conditions, wherebythe nucleic acid binding protein encoded by the DNA is expressed. Thecomposition of suitable media is known to those in the art, so that theycan be readily prepared. Suitable culturing media are also commerciallyavailable.

[0081] Nucleic acid binding molecules according to the invention may beemployed in a wide variety of applications, including diagnostics and asresearch tools. Advantageously, they may be employed as diagnostic toolsfor identifying the presence of nucleic acid molecules in a complexmixture.

[0082] Zinc finger domains may be made by methods described and/orreferred to herein. For example, said zinc linger DNA binding domainsmay be made as discussed in the examples, or as described in one or moreof WO96/06166, WO98/53058, WO98/53057, or WO/98/53060.

Telomeres, G-quadruplexes and G-quartets

[0083] Telomeres comprise highly conserved DNA repeat sequences,associated with proteins, found at the ends of chromosomes in nearly alleukaryotes. They are widely studied because of their important roles inmaintaining chromosome stability and in mediating normal chromosomesegregation in mitosis and meiosis (Rhodes, D.. & Giraldo, R. (1995)Curr Opin Str Biol 5, 311-322.).

[0084] Because of their potential importance, G-quadruplexes have beenextensively characterised in terms of structure, polymorphism, ionselectivity, stability and folding kinetics [reviewed in (Williamson, J.R. (1994) Annual Review Of Biophysics and Biomolecular Structure 23,703-730)].

[0085] Telomeric DNA sequences contain characteristic guanine-richrepeats of the form d(T₁₋₃-(T/A)-G₃₋₄)_(n) [reviewed in (Blackburn, E.H., & Szostak, J. W. (1984) Annual Review Of Biochemistry 53,163-194.)]. These sequences form G-quadruplex secondary structures invitro at physiological salt concentrations (K⁺ or Na⁺) and it has beenproposed that such structures may be of biological significance in vivo.It has been suggested that inter-telomeric G-quadruplexes may determinethe correct association of homologous chromosomes in different stages ofthe cell cycle (Sen, D., & Gilbert, W. (1988) Nature 334, 364-366.Sundquist, W. I., & Klug. A. (1989) Nature 342, 825-829. Williamson, J.R., Raghuraman, M. K., & Cech, T. R. (1989) Cell 59, 871-880.). Morerecently, it has been suggested that the G-quadruplex conformation ofsingle stranded telomeric DNA may be important to the mechanism andregulation of telomerase-mediated telomere extension (Salazar M,Thompson B D, Kerwin S M, Hurley L H. (1996) Biochemistry35(50):16110-5. Sun. D., LopezGuajardo, C. C., Quada, J., Hurley, L. H.,& VonHoff, D. D. (1999) Biochemistry 38, 4037-4044.). Furthermore,G-quadruplexes have emerged as a molecular target for therapeuticsparticularly in cancer research (Sun D, Thompson B, Cathers B E, SalazarM, Kerwin S M, Trent J O, Jenkins T C, Neidle S, Hurley L H (1997) J MedChem July 4; 40(14):2113-6. Perry P J, Reszka A P, Wood A A. Read M A,Gowan S M, Dosanjh H S, Trent J O, Jenkins T C, Kelland L R, Neidle S.(1998) J. Med Chem. 41(24):4873-84.).

[0086] Several naturally occurring proteins with affinity forG-quadruplexes have been reported (Wellinger, R. J., & Sen, D. (1997)European Journal of Cancer 33, 735-749.), although there are problemsassociated with their use as diagnostic or therapeutic probes. Mostexamples, such as a recently reported DNA-binding autoantibody (Brown,B. A., Li, Y. Q., Brown, J. C., Hardin, C. C., Roberts, J. F., Pelsue,S. C., & Shultz, L. D. (1998) Biochemistry 37, 16325-16337.), have onlymoderate binding affinities and discriminate weakly between duplex andquadruplex DNA. Naturally occurring high-affinity telomere-bindingproteins also appear unable to discriminate these structures. Forexample. Saccharomyces cerevisiae RAP1 (Giraldo, R., & Rhodes, D. (1994)EMBO J 13. 2411-2420.) has distinct but inseparable domains for bindingquadruplexes and double stranded DNA.

[0087] Prior art telomere-binding proteins have only moderate bindingaffinities and/or discriminate weakly between duplex and quadruplex DNA.

[0088] The molecules of the present invention may be used as probes forthe presence of G-quadruplex structures, both in vitro and in vivo.

[0089] The the present invention facilitates ELISA-based detection oftelomerase activity. This detection system is rapid, easily automatedwith liquid handling robotics and avoids the need to use radioactivity.This contrasts with prior art telomerase assays such as thecommercially-available ‘TRAP’ assay.

[0090] Telomere-binding molecules according to the present invention maybe used to target chromosome ends and deliver effector activity, forexample using fusions with other peptides or enzymes.

[0091] It is envisaged that the present invention may be of use indiverse areas, including for example one or more of the following;diagnostics, assays, elisa testing, probe production, genomics studiessuch as pharmacogenomics, therapeutic applications such as study orconstruction of disease model(s), drug design, peptide/protein research,the construction or exploitation of research tools such as molecularmarker(s) and similar reagents, as well as use in screening such asusing technology, cellular or in vitro assay(s), molecular detection aswell as target identification or validation.

[0092] The present invention may also be of use in the study and/ortreatment of metabolic disorders, or cancer.

[0093] The present invention facilitates the construction of ELISA-baseddiagnostic kits for the detection of telomerase activity. These assaysare rapid, easily automated with liquid handling robotics and avoid theneed to use radioactivity, in contrast to prior art technologies such asthe ‘TRAP’ assay.

[0094] The present invention encompasses the development of probes forexamining G-quartet formation in vivo.

[0095] Telomere-binding molecules according to the invention may be usedto target chromosome ends and to deliver effector activity in the formof fusions with other peptides or enzymes. Therapeutic applications ofthe invention include those associated with the role(s) of telomeres inageing and/or cancer.

[0096] Telomere-binding molecules according to the invention may affecttelomerase activity and may be used as, or in conjunction with,inhibitors of this enzyme, the activity of which is associated with cellimmortalisation and cancer.

[0097] Zinc finger protein molecules according to the invention may beselected from a phage display library to bind G-quadruplex DNAstructures of single stranded human telomeric sequences with selectivityand high affinity. Advantageously, these zinc fingers have no detectableaffinity for a duplex DNA made up of the Htelo sequence and itscomplementary strand. These molecules represent a new class ofDNA-binding zinc lingers and have utility for both study and explorationof the molecules themselves, and of therapeutics and assays, in additionto their utility as in vitro or in vivo molecular probes to explorepossible mechanisms of inhibition and regulation of telomerase-mediatedtelomere extension. The widespread conservation of G-quadruplex-formingsequences at chromosome ends means that the molecules according to theinvention will find utility in a wide range of biological systems.

[0098] Since in vitro diagnostic methods for detecting G-quadruplexes,such as circular dichroism (Giraldo, R., Suzuki, M., Chapman, L., &Rhodes, D. (1994) Proc Natl Acad Sci 91, 7658-7662.) and dimethylsulphate protection (Sen, D., & Gilbert. W. (1992) Methods In Enzymology211, 191-199.), cannot be carried out in living cells, the invention isuseful in relation to derivatives (e.g. fluorescent derivatives) ofthese zinc fingers which may reveal the presence, location and relevanceof these telomeric structures in vivo.

[0099] Molecules according to the present invention are useful in thebinding of non-conventional nucleic acid structures. Examples of suchstructures include non-Watson-Crick base paired DNA, for exampleHoogsteen base paired DNA or other variants. Furthermore,non-conventional DNA structures include non-double helical DNAconformations.

Fusions

[0100] According to a further aspect, the invention provides a nucleicacid binding polypeptide capable of binding to telomeric, G-quadruplex,or G-quartet nucleic acid wherein said polypeptide comprises a nucleicacid binding domain and one or more further domain(s) joined thereto.Said domains may be joined by any suitable means known in the art, suchas by conjugation, fusion, or other suitable method. Preferably, saiddomains are comprised by a single polypeptide fusion protein. Such anucleic acid binding polypeptide may comprise nucleic acid bindingdomains linked by at least one flexible linker, one or more domainslinked by at least one structured linker, or both.

[0101] According to a further aspect, the invention provides a nucleicacid binding polypeptide comprising a repressor domain and one or morenucleic acid binding domains. The repressor domain is preferably atranscriptional repressor domain selected from the group consisting of:a KRAB-A domain, an engrailed domain and a snag domain.

[0102] The nucleic acid binding polypeptides according to our inventionmay be linked to one or more transcriptional effector domains, such asan activation domain or a repressor domain. Examples of transcriptionalactivation domains include the VP16 and VP64 transactivation domains ofHerpes Simplex Virus. Alternative transactivation domains are variousand include the maize C1 transactivation domain sequence (Sainz et al.,1997, Mol. Cell. Biol. 17: 115-22) and P1 (Goff et al., 1992, Genes Dev.6: 864-75; Estruch et al., 1994, Nucleic Acids Res. 22: 3983-89) and anumber of other domains that have been reported from plants (see Estruchet al., 1994, ibid).

[0103] Instead of incorporating a transactivator of gene expression, arepressor of gene expression can be fused to the nucleic acid bindingpolypeptide and used to down regulate the expression of a genecontiguous or incorporating the nucleic acid binding polypeptide targetsequence. Such repressors are known in the art and include, for example,the KRAB-A domain (Moosmann et al., Biol. Chem. 378: 669-677 (1997)),the KRAB domain from human KOX1 protein (Margolin et al., PNAS91:4509-4513 (1994)), the engrailed domain (Han et al., Embo J. 12:2723-2733 (1993)) and the snag domain (Grimes et al., Mol Cell. Biol.16: 6263-6272 (1996)). These can be used alone or in combination todown-regulate gene expression.

[0104] Molecules according to the invention comprising zinc fingerproteins may be fused to transcriptional repression domains such as theKruppel-associated box (KRAB) domain to form powerful repressors. Thesefusions are known to repress expression of a reporter gene even whenbound to sites a few kilobase pairs upstream from the promoter of thegene (Margolin et al., 1994, PNAS USA 91, 4509-4513).

[0105] Nucleic acid binding molecules according to the invention maycomprise tag sequences to facilitate studies and/or preparation of suchmolecules. Tag sequences may include flag-tag, myc-tag, 6his-tag or anyother suitable tag known in the art.

[0106] Advantageously, molecules according to the invention may be usedin combination. Use in combination includes both fusion of moleculesinto a single polypeptide as well as use of two or more discretepolypeptide molecules in solution.

[0107] The invention thus relates to the manipulation of telomericstructures using zinc finger peptides and derivative fusion proteinsaccording to the invention. Examples of such manipulation include simplebinding, modification eg. methylation, cleavage eg. by nuclease action,or other chemical or physical modification.

[0108] Further fusion proteins according to the invention are describedherein, for example in the following section.

Pharmaceuticals

[0109] Moreover, the invention provides therapeutic agents and methodsof therapy involving use of nucleic acid binding proteins as describedherein. In particular, the invention provides the use of polypeptidefusions comprising an integrase, such as a viral integrase, and anucleic acid binding protein according to the invention to targetnucleic acid sequences in vivo (Bushman, (1994) PNAS (USA)91:9233-9237). In gene therapy applications, the method may be appliedto the delivery of functional genes into defective genes, or thedelivery of nonsense nucleic acid in order to disrupt undesired nucleicacid. Alternatively, genes may be delivered to known, repetitivestretches of nucleic acid, such as centromeres, together with anactivating sequence such as an LCR. This would represent a route to thesafe and predictable incorporation of nucleic acid into the genome.

[0110] In conventional therapeutic applications, nucleic acid bindingproteins according to the invention may be used to specifically knockout cell having mutant vital proteins. For example, if cells with mutantras are targeted, they will be destroyed because ras is essential tocellular survival. Alternatively, the action of transcription factorsmay be modulated, preferably reduced, by administering to the cellagents which bind to the binding site specific for the transcriptionfactor. For example, the activity of HIV tat may be reduced by bindingproteins specific for HIV TAR.

[0111] Moreover, binding proteins according to the invention may becoupled to toxic molecules, such as nucleases, which are capable ofcausing irreversible nucleic acid damage and cell death. Such nucleasesinclude restriction endonuclease domains, non-specific nucleases such asDNAse, RNAse or similar enzymatic acticvities. Such agents are capableof selectively destroying cells which comprise a mutation in theirendogenous nucleic acid.

[0112] Nucleic acid binding proteins and derivatives thereof as setforth above may also be applied to the treatment of infections and thelike in the form of organism-specific antibiotic or antiviral drugs. Insuch applications, the binding proteins may be coupled to a nuclease orother nuclear toxin and targeted specifically to the nucleic acids ofmicroorganisms.

[0113] The invention likewise relates to pharmaceutical preparationswhich contain the compounds according to the invention orpharmaceutically acceptable salts thereof as active ingredients, and toprocesses for their preparation.

[0114] The pharmaceutical preparations according to the invention whichcontain the compound according to the invention or pharmaceuticallyacceptable salts thereof are those for enteral, such as oral,furthermore rectal, and parenteral administration to (a) warm-bloodedanimal(s), the pharmacological active ingredient being present on itsown or together with a pharmaceutically acceptable carrier. The dailydose of the active ingredient depends on the age and the individualcondition and also on the manner of administration.

[0115] The novel pharmaceutical preparations contain, for example, fromabout 10% to about 80%, preferably from about 20% to about 60%, of theactive ingredient. Pharmaceutical preparations according to theinvention for enteral or parenteral administration are, for example,those in unit dose forms, such as sugar-coated tablets, tablets,capsules or suppositories, and furthermore ampoules. These are preparedin a manner known per se, for example by means of conventional mixing,granulating, sugar-coating, dissolving or lyophilising processes. Thus,pharmaceutical preparations for oral use can be obtained by combiningthe active ingredient with solid carriers, if desired granulating amixture obtained, and processing the mixture or granules, if desired ornecessary, after addition of suitable excipients to give tablets orsugar-coated tablet cores.

[0116] Suitable carriers are, in particular, fillers, such as sugars,for example lactose, sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example tricalcium phosphateor calcium hydrogen phosphate, furthermore binders, such as starchpaste, using, for example, corn, wheat, rice or potato starch, gelatin,tragacanth, methylcellulose and/or polyvinylpyrrolidone, if desired,disintegrants, such as the abovementioned starches, furthermorecarboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginicacid or a salt thereof, such as sodium alginate; auxiliaries areprimarily glidants, flow-regulators and lubricants, for example silicicacid, talc, stearic acid or salts thereof, such as magnesium or calciumstearate, and/or polyethylene glycol. Sugar-coated tablet cores areprovided with suitable coatings which, if desired, are resistant togastric juice, using, inter alia, concentrated sugar solutions which, ifdesired, contain gum arabic, talc, polyvinylpyrrolidone, polyethyleneglycol and/or titanium dioxide, coating solutions in suitable organicsolvents or solvent mixtures or, for the preparation of gastricjuice-resistant coatings, solutions of suitable cellulose preparations,such as acetylcellulose phthalate or hydroxypropylmethylcellulosephthalate. Colorants or pigments, for example to identify or to indicatedifferent doses of active ingredient, may be added to the tablets orsugar-coated tablet coatings.

[0117] Other orally utilisable pharmaceutical preparations are hardgelatin capsules, and also soft closed capsules made of gelatin and aplasticiser, such as glycerol or sorbitol. The hard gelatin capsules maycontain the active ingredient in the form of granules, for example in amixture with fillers, such as lactose, binders, such as starches, and/orlubricants, such as talc or magnesium stearate, and, if desired,stabilisers. In soft capsules, the active ingredient is preferablydissolved or suspended in suitable liquids, such as fatty oils, paraffinoil or liquid polyethylene glycols, it also being possible to addstabilisers.

[0118] Suitable rectally utilisable pharmaceutical preparations are, forexample, suppositories, which consist of a combination of the activeingredient with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. Furthermore, gelatin rectalcapsules which contain a combination of the active ingredient with abase substance may also be used. Suitable base substances are, forexample, liquid triglycerides, polyethylene glycols or paraffinhydrocarbons.

[0119] Suitable preparations for parenteral administration are primarilyaqueous solutions of an active ingredient in water-soluble form, forexample a water-soluble salt, and furthermore suspensions of the activeingredient, such as appropriate oily injection suspensions, usingsuitable lipophilic solvents or vehicles, such as fatty oils, forexample sesame oil, or synthetic fatty acid esters, for example ethyloleate or triglycerides, or aqueous injection suspensions which containviscosity-increasing substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and, if necessary, alsostabilisers.

[0120] The dose of the active ingredient depends on the warm-bloodedanimal species, the age and the individual condition and on the mannerof administration. In the normal case, an approximate daily dose ofabout 10 mg to about 250 mg is to be estimated in the case of oraladministration for a patient weighing approximately 75 kg.

[0121] The present invention has advantages over existing technologywhich include but are not limited to increased speed and sensitivitywhen using amplifying ELISA signal, removal of the need for runningelectrophoretic gels, and alleviation of the need to use radioactivelabelling. Furthermore, the systems of the present invention can beadvantageously automated using liquid-handling robotics, resulting inhigh efficiency and labour-saving.

[0122] Without wishing to be bound by theory, the nature of the zincfinger-G-quadruplex interactions is likely to be quite distinct fromknown zinc finger-duplex DNA interactions.

[0123] The invention will now be described by way of Example, withreference to the following figures.

BRIEF DESCRIPTION OF THE FIGURES

[0124]FIG. 1a shows schematic representation of the Zif268 DNA-bindingdomain, indicating its three zinc finger helices (F1, F2 and F3). Thecircled numbers represent the key amino acid residues that interact withduplex DNA (relative to the first position of the α-helix, position +1).

[0125]FIG. 1b shows amino acids included in the phage display libraryused in this study. Amino acid residues in the helical regions offingers 1-3 (F1-F3) are shown in single letter code, numbered relativeto the first helical position (position +1). Note that libraryconstruction involved cloning a subset of the possible combinationsshown above, although these clones were pre-enriched for DNA-bindingpotential (See below).

[0126]FIG. 2a shows DMS methylation protection analysis of Htelo.End-labelled ³²P-Htelo was annealed in KCl or NaCl at the indicatedconcentrations. Each sample was incubated with DMS for 5 minutes andthen cleaved with piperidine. Methylation protection patterns,indicative of G-quadruplex formation, appear after resolution of thecleaved fragments on a 20% polyacrylamide gel. The Tris control laneindicates the reference (non-quadruplex) methylation cleavage pattern ofHtelo in the absence of Na⁺ or K⁺.

[0127]FIG. 2b shows schematic representation of an exemplary isoform ofan intramolecular antiparallel G-quadruplex formed by Htelo. Guanines inthe G-quartet core are labelled in shaded circles with darker shadingindicating a relatively higher amount of cleavage, as observed in theDMS methylation protection analysis. (Note that the structure shown isonly one possible isoform and that other ‘semi-parallel’ conformation(s)such as one comprising a pair of parallel ‘up’ strands, facing a pair ofparallel ‘down’ strands created by ‘crossing-over’ of the two top ‘TTA”sequences in the figure may also be stable form(s) of Htelo.)

[0128]FIG. 3 shows peptide sequences of the zinc finger helical domainsof the four proteins Gq1-4, obtained after three rounds of selection.Amino acid residues in Fingers 1-3 (F1-F3) are shown in single lettercode, numbered relative to the first helical position (position +1). Thezinc finger helices of the wild-type Zif268 DNA-binding domain are alsoshown for comparison.

[0129]FIG. 4 shows apparent equilibrium binding curves for protein Gq1binding to single-stranded DNA sequences, and to the Htelo duplexsequence, as measured by phage ELISA. All ELISA procedures were carriedout in the presence of 150 mM K⁺, to stabilise C-quadruplexes.

[0130]FIG. 5a shows gel mobility shift assay of Gq1* binding to Htelo.The analysis was carried out in 8% non-denaturing polyacrylamide gel at4° C. The DNA concentration is fixed at 1 nM while the amount of proteinadded to the binding reaction is varied as follows: 800 nM (lane 1), 400nM (lane 2), 200 nM (lane 3), 100 nM (lane 4), 50 nM (lane 5), 25 nM(lane 6), 12.5 nM (lane 7) and 0 nM (lane 8)

[0131]FIG. 5b shows apparent equilibrium binding curve obtained bycalculating the fraction of Htelo bound at varying Gq1* concentrations(Imagequant software). The binding constant was determined by fitting tothe equation Ø=[P]/{K_(d)+[P]} as described in the Examples.

[0132]FIG. 6 shows DMS methylation protection analysis of Htelo in thepresence of Gq1* protein. Htelo was annealed in 100 mM K⁺ or 50 mMTris-HCl, and methylation protection patterns were obtained in thepresence or absence of 200nM Gq1* (ie. 200 nM Gq1*—a concentrationgiving approximately full shift). DNA concentration is 1 nM. Each samplewas incubated with DMS for 5 min. Fragments formed by piperidinecleavage of methylated guanines, were resolved on a 20% polyacrylamidegel. Lane 1: methylation pattern of Htelo in the presence of 100 mM K⁺;Lane 2: reference methylation pattern of Htelo in the absence of K⁺;Lane 3: methylation pattern of Htelo in the presence of 100 mM K⁺ andincubation with 200 nM Gq1*; Lane 4: methylation pattern in the absenceof K⁺, incubated with 200 nM Gq1*.

[0133]FIG. 7 shows Table 1 which shows apparent ELISA dissociationconstants (Kd^(E)) of the phage-displayed zinc finger peptide. Gq1, forvariants of the Htelo DNA sequence. ELISAs from which binding is too lowto determine Kd are denoted by a dash (-).

EXAMPLES Example 1 Production of Molecules Binding G-quadruplexStructures

[0134] In this Example, DNA-binding proteins of the zinc finger familyare engineered to bind specifically to a telomeric G-quadruplex nucleicacid structure.

[0135] A zinc finger library is screened for molecules that bind to anoligonucleotide containing the human telomeric repeat sequence in theG-quadruplex conformation. The selected molecular clones exhibit aminoacid homologies (consensus sequences). Without wishing to be bound bytheory, this suggests that the molecules have analogous modes ofbinding. Binding is both sequence-dependent and structure-specific. Thisis the first example of a designed molecule that binds to G-quadruplexDNA. Further, this represents a new type of binding interaction for azinc finger protein molecule.

G-quadruplex DNA Ligand Preparation

[0136] It has been previously reported that the human telomeric sequence(5′-GTTAGG-3′)_(n) forms G-quadruplex structures in vitro(Balagurumoorthy, P., Brahmachari, S. K., Mohanty, D., Bansal, M., &Sasisekharan, V. (1992) Nucleic Acids Research 20, 4061-4067.Balagurumoorthy, P., & Brahmachari, S. K. (1994) Journal Of BiologicalChemistry 269, 21858-21869. Fletcher, T. M., Sun, D. K., Salazar, M., &Hurley, L. H. (1998) Biochemistry 37, 5536-5541.). The five repeattelomeric oligonucleotide sequence (5′-GTTAGG-3′)₅ (Htelo) is employedas the ligand for affinity selection of phage herein.

[0137] Synthesised oligonucleotides (Oswel Ltd.) are purified byfractionation in denaturing polyacrylamide-urea gels, recovered byelution and desalted further using Waters sep-Pack C-18 cartridges withfinal elution in 25 mM Tris, pH 7.5 as described by Giraldo et al.(Giraldo, R., & Rhodes, D. (1994) EMBO J 13, 2411-2420.). The sequence5′-biotin-GGTTAG GGTTAG GGTTAG GGTTAG GGTTAG-3′ (‘Biotin-Htelo’) isprepared for the phage selection experiments and the unbiotinylatedsequence (‘Htelo’) is used for gel mobility shift and DMS protectionexperiments.

[0138] Oligonucleotides are then annealed for quadruplex formation, andsubsequently used for ELISA and/or gel assays (see below).Oligonucleotides are diluted to 10 pmol/μl in 25 mM Tris (pH 7.5) orphosphate-buffered KCl or NaCl (pH 7.5) with cation concentrationsranging from 25 mM to 150 mM. Annealing or quadruplex formation iscarried out by heating samples to 95° C. on a thermal heating block, andcooling to 4° C. at a rate of 2° C./min. The double stranded DNA (dsHtelo) is made by primer extension with the Klenow fragment of DNApolymerase.

[0139] Structures formed by human telomeric sequences may be analysedusing dimethyl sulphate protection analysis to determine the existenceof G-quadruplexes therein. To confirm that Htelo is folded into aG-quadruplex in the presence of sodium and potassium ions, a dimethylsulphate (DMS) protection assay is carried out (Sundquist, W. I., &Klug, A. (1989) Nature 342, 825-829.). G-quadruplex formation involvesHoogsteen-type base pairing of guanines which protects N-7 of guanineagainst methylation on exposure to the potent methylating agent DMS.Subsequent cleavage of the DNA backbone at methylated guanines can bemediated by heating in aqueous piperidine (Maxam, A. M., & Gilbert, W.(1980) Methods Enzymol. 65, 499-560.).

[0140] The resulting gel pattern (see for example FIG. 2a) clearly showsthat the critical guanines of Htelo are almost completely protected fromcleavage, at K⁺ or Na⁺ concentrations above 100 mM, as compared to aTris-HCl buffer control. Non-denaturing gels confirm that these foldedforms are of a single species and therefore antiparallel intramolecularG-quadruplexes, ie. similar to the structure illustrated in FIG. 2b.Intermolecular G-quadruplexes are not observed in detectable amountsunder these conditions. Without wishing to be bound by theory, this isprobably because of their slow folding kinetics and/or because of therelatively low concentrations of DNA used which are likely to promoteintramolecular G-quadruplex formation (Hardin C C, Henderson E, WatsonT. Prosser J K (1991) Biochemistry May 7; 30(18):4460-72).

[0141] A zinc finger phage display library is constructed specificallyto select candidates that bind human telomeric DNA sequences, underconditions that promote G-quadruplex formation. The library is made upof zinc fingers with selectively randomised residues, biased for dsDNAbinding potential (Choo, Y., & Klug, A. (1994) Proc. Natl. Acad. Sci.U.S.A. 91, 11163-11167. Isalan, M., Klug, A., & Choo, Y. (1998)Biochemistry 37, 12026-12033). Similar libraries have been extensivelycharacterised, both biochemically and structurally, but only in theircapacity to bind duplex DNA sequences in the major groove. (Choo, Y., &Klug. A. (1997) Curr. Opin. Str. Biol. 7, 117-125. Choo, Y., & Isalan,M. D. (2000) Current Opinion in Structural Biology 10).

[0142] Because of practicalities of library handling, a complementarysub-library strategy is employed. Consequently, two completesub-libraries are constructed and enriched for DNA-binding potential byselection against randomised dsDNA sequences (see below). The resultingclones are recombined in vitro to make a library containingrandomisations over all three fingers.

Construction of Phage Display Library

[0143] A phage display library is constructed, based on the three-fingerDNA-binding domain of Zif268, whose structure is well characterised(Elrod-Erickson, M., Rould, M. A., Nekludova, L., & Pabo, C. O. (1996)Structure 4, 1171-1180. Pavletich, N. P., & Pabo, C. O. (1991) Science252, 809-817.).

[0144] A zinc finger DNA-binding domain library is constructedcomprising the amino acid framework of wild-type Zif268, but containingrandomisations in amino acid positions over all three fingers (see FIG.1). Due to the practicalities of library cloning (ie. working with about˜10⁶-10⁷ transformants), the final library is advantageously constructedfrom two complementary sub-libraries: Sub-library-1 containsrandomisations in F1 (−1→6) and F2 (−1→3). Conversely, sub-library-2contains randomisations in F2 (3→6) and F3 (−1→6). In bothsub-libraries, the non-randomised regions retain the wild-type Zif268framework.

[0145] The genes for each sub-library are assembled from synthetic DNAoligonucleotides by directional end-to-end ligation using shortcomplementary DNA linkers. The oligonucleotides contain selectivelyrandomised codons, encoding a subset of the 20 amino acids, in theappropriate positions within the zinc fingers. Assembled constructs areamplified by PCR using primers containing Not I and Sfi I restrictionsites, digested with the above endonucleases to produce cloningoverhangs, and ligated into similarly prepared vector Fd-Tet-SN (Choo,Y., & Klug, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11163-11167.)Electrocompetent E. coli TG1 cells arc transformed with the recombinantvector and plated onto TYE medium (1.5% (w/v) agar, 1% (w/v)Bactotryptone, 0.5% (w/v) Bactoyeast extract, 0.8% (w/v) NaCl)containing 15 μg/ml tetracycline.

[0146] The sub-libraries are enriched for DNA-binding members byselecting against random DNA-sequences.

[0147] The 3-finger phage library is screened with 5′-biotin-(GGTTAG)₅(Biotin-Htelo) which has been annealed in a phosphate-buffered solutioncontaining 150 mM potassium ions then immobilised on streptavidin tubes.These salt conditions are maintained throughout the selection protocolto help maintain the structural integrity of the G-quadruplex.

[0148] Phage selections are performed as follows:

[0149] Tetracycline resistant library colonies of E. coli TG1 cells aretransferred from plates into 2×TY medium (16 g/litre Bactotryptone, 10g/litre Bactoycast extract, 5 g/litre NaCl) containing 50 μM ZnCl₂ and15 μg/ml tetracycline, and cultured overnight at 30° C. in a shakingincubator. Cleared culture supernatant containing phage particles isobtained by centrifuging at 300 g for 5 minutes.

[0150] For the first rounds of selection, appropriate quantities ofbiotinylated DNA target site are immobilised on streptavidin-coatedtubes (Roche) in 50 μl phosphate buffer (pH 7.4) containing 50 μM ZnCl₂and 150 mM KCl for 30 minutes at room temperature. Bacterial culturesupernatant containing phage is diluted 1:10 in selection buffer(phosphate buffer pH 7.4 with 150 mM KCl) containing 50 μM ZnCl₂, 2%(w/v) fat-free dried milk (Marvel), 1% (v/v) Tween, 20 μg/ml sonicatedsalmon sperm DNA), and 1 ml is applied to each tube. After 1 hour at 20°C., the tubes are emptied and washed 20 times with selection buffercontaining 50 μM ZnCl₂, 2% (w/v) fat-free dried milk (Marvel) and 1%(v/v) Tween.

[0151] Retained phage are eluted in 0.1 M trimethylamine and neutralisedwith an equal volume of I M Tris-HCl (pH 7.4). Logarithmic-phase E. coliTG1 are infected with eluted phage, and cultured overnight at 30° C. in2×TY medium containing 50 μM ZnCl₂ and 15 μg/ml tetracycline, to amplifyphage for subsequent rounds of selection.

[0152] For enrichment of the sub-libraries 1 and 2, 50 pmol ofbiotinylated semi-random oligonucleotides of the form

5′-TATANNNNNNNGGCGTGTCACAGTCAGCTTCAACGTC-3′

and

5′-TATGTGCGGNNNNNNNTCACAGTCAGTCCACACGTC-3′,

[0153] respectively, are used in selection round 1. These amounts arereduced to 20 pmol and 10 pmol in rounds 2 and 3.

[0154] The heterogeneous genes from the selected clones are recovered byPCR and recombined via a DdeI site, present in the sequence coding forpositions +4 and +5 in F2 of both libraries (see WO98/53057).Recombinants are then re-cloned into phage vector, as described above.Ultimately, 3×10⁶ selection-enriched library members are obtained,containing randomisations over all 3 zinc fingers.

[0155] For selections against Biotin-Htelo, using the full recombinedlibrary, 100 pmol of the pre-annealed oligonucleotide is immobilised onstreptavidin-coated tubes in the first round. In rounds 2 and 3,selection pressure is increased by reducing the amount of target site to50 pmol and 1 pmol, respectively. In these rounds, 50 pmol of duplex and50 pmol single stranded competitor oligonucleotides are also added ofthe form: 5′-TATANNNNNNNNNNNNNTCACAGTCAGTCCACACGTC-3′. After 3 rounds ofselection, E. coli TG1 infected with selected phage are plated.Individual colonies are picked and used to prepare phage for ELISAassays and DNA sequencing.

[0156] After three rounds of selection, four different zinc fingerclones are recovered and individually screened for binding toimmobilised Biotin-Htelo by an ELISA assay (Choo, Y., & Klug, A. (1994)Proc. Natl. Natl. Acad. Sci. U.S.A. 91, 11163-11167.).

[0157] The four isolated clones (Gq1-4) are sequenced. The codingsequence of individual zinc finger clones is amplified by PCR from phagesamples. PCR products are sequenced manually using Thermo Sequenasecycle sequencing (Amersham Life Science).

[0158] The aligned sequences are shown in FIG. 3. The clones appear tohave a significant degree of sequence similarity which is indicative ofa successful selection process and suggests analogous functions for eachclone. Control binding assays confirm that neither the phage nor theZif268 are able to bind to Biotin-Htelo.

[0159] The sequence composition of the zinc finger helices from Zif268is also shown for comparison in FIG. 3. The palindromic chargedistributions of the selected zinc fingers are very different to that ofZif268. It is interesting to note that finger 2 (F2) of Gq1-4 have eachselected negatively charged acidic sidechains (Asp or Glu) particularlyin positions labelled −1 3 and 6 (FIG. 3). This pattern is unusual forDNA-binding zinc fingers as negatively charged residues are expected torepel the surface of the phosphodiester backbone. Without wishing to hebound by theory, it is possible that these acidic residues interact withguanine —NH groups which line all four grooves of an antiparallelG-quadruplex's helical core.

[0160] Zinc finger protein molecule(s) selected from this libraryaccording to the invention bind to single stranded human telomeric DNAwith an affinity comparable to that of natural transcription factors.

[0161] There is strong discrimination between the double-stranded formof the same sequence and single-stranded variants.

Example 2 Molecules According to the Invention Selectively BindG-quadruplex DNA

[0162] The nucleic acid binding properties of zinc finger moleculesproduced according to the invention may be analysed.

[0163] Characterisation of the binding properties of molecules Gq1-4(see Example 1) shows that they do indeed behave very similarly.Therefore, only one phage clone (Gq1) is used to explore the bindingspecificity in more detail in this Example.

[0164] Phage ELISA is performed using analogues of the Biotin-Htelooligonucleotide which contain adenine or inosine substitutions forcritical guanine residues which are important for G-quadruplex formation(see Table 1). Although adenine and inosine are structurally related toguanine, both destabilise G-quadruplex formation (Williamson, J. R.,Raghuraman, M. K., & Cech, T. R. (1989) Cell 59, 871-880.). The adeninesubstitution leads to a hydrogen bonding arrangement that isincompatible with G-quartet formation, while inosine lacks an N-2exocyclic amino group required for fully stabilising such structures.

[0165] The phage ELISA used herein is adapted from previous assays(Choo. Y., & Klug, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91,11163-11167.). 5′-biotinylated DNA sites are added tostreptavidin-coated ELISA wells (Boehringer-Mannheim) in 50 mM potassiumphosphate buffer (pH 7.5) containing 100 mM potassium chloride and 50 μMZinc chloride (K/Zn buffer). Phage solution [overnight bacterial culturesupernatant diluted 2:10 in K/Zn buffer containing 2% (w/v) fat-freedried milk (Marvel), 1% (v/v) Tween and 20 μg/ml sonicated salmon spermDNA] was applied to each well (50 μl/well). The phage are allowed tobind for 1 hour at 20° C. Unbound phage are removed by washing 6 timeswith K/Zn buffer containing 1% (v/v) Tween, and then 3 times with K/Znbuffer. Bound phage are detected by ELISA using horseradishperoxidase-conjugated anti-M13 IgG (Pharmacia Biotech), and thecolorimetric signal was quantified using BIO KINETICS READER EL 340(Bio-Tck Instruments).

[0166] Under the binding assay conditions used (150 mM K⁺), Gq1 has anapparent ELISA dissociation constant (K_(d) ^(E)) of 26 nM forBiotin-Htelo (eg. see Table 1, FIG. 4). No significant binding of Gq1 isobserved for any of the guanine-substituted analogues employed,suggesting Gq1 is highly structure-specific for G-quadruplex nucleicacid.

[0167] A double stranded Htelo oligonucleotide ligand is made by DNApolymerase primer extension of the C-rich complementary sequence ofHtelo. This complex is also analysed for binding of G1q by ELISA andexhibits no significant binding (Table 1). Therefore although Gq1 isspecific for the Htelo sequence, it cannot bind this sequence in thedouble-helical conformation.

[0168] Thus, molecules according to the invention bind G-quadruplexnucleic acid in a highly structure-specific manner.

[0169] The characteristics of this Example of a molecule according tothe invention are further investigated using electromobility shiftassays on G-quadruplex DNAs and DMS protection of the DNA-proteincomplex.

[0170] To explore the nature of the Gq1-Htelo complex in more detail,the gene encoding Gq1 is cloned and overexpressed as aglutathione-S-transferase fusion protein (‘Gq1*’) in E. coli (ChittendenT, Livingston D M, Kaelin W G Jr (1991) Cell June 14; 65(6):1073-82;Smith D B, Johnson K S (1988) Gene July 15; 67(l):31-40).

[0171] The zinc finger gene is amplified by PCR, using 1 μl overnightbacterial culture supernatant (containing phage) as template. Theprimers introduced BamHI sites for ligation into vector pGEX-3X(Amersham-Pharmacia). The resulting construct (Gq1*), coding for GSTfused in frame with C-terminal zinc fingers, is cloned in E. coli TG1and verified by DNA sequencing. Fusion protein expression is thencarried out in E. coli BL21 DE3. Gq1* is purified from bacterial lysatesby affinity chromatography using Glutathione Sepharose 4 Fast Flow(Pharmacia Biotech).

[0172] The eluted protein appears as a single band of >95 % totalprotein on a protein gel, and corresponds to the expected molecularweight of 37 kD.

[0173] The complex between Gq1* and oligonucleotide Htelo is studied bynon-denaturing gel mobility shift analysis (Cann J R (1989) J Biol ChemOctober 15; 264(29):17032-40. Garner M M, Revzin A (1981) Nucleic AcidsRes July 10; 9(13):3047-60) as follows;

Gel Mobility Shift Analysis

[0174] Binding reactions are performed in a final volume of 10 μl, using10 fmol of labelled oligonucleotide and various amounts of purified Gq1*in binding buffer: 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 6%glycerol, 100 μg/ml BSA, 1 μg/ml calf thymus DNA, 50 μM ZnCl₂ and KCl to150 mM. Binding reactions are carried out at room temperature for 1hour. The samples are loaded on a 8% polyacrylamide(acrylamide:bisacrylamide=33:1) non-denaturing gel. The buffer in thegel and for electrophoresis is 0.5×TB buffer (Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor.).Electrophoresis is performed at 15 V/cm, for 2 hours, at 4° C. The gelsare exposed in a phosphorimager cassette and imaged (Model 425EPhosphorImager; Molecular Dynamics, Inc). The bands are quantified usingImagequant software. The fraction of DNA that is bound and free isdetermined after normalisation by summing the total number of counts ineach lane (Senear D F, Brenowitz M (1991) J Biol Chem July 25;266(21):13661-71). To minimise any error due to perturbation of theequilibrium under electrophoretic conditions, the fraction of free DNAis measured at various protein concentrations rather than measuring theamount of complex formed (Cann J R (1989) J Biol Chem October 15;264(29):17032-40; Garner M M, Revzin A (1981) Nucleic Acids Res July 10;9(13):3047-60). The data is plotted as Ø (1-fraction of free DNA) vsprotein concentration to determine the K_(d), which is equal to theprotein concentration at which half the free DNA is bound. Equilibriumdissociation constants (K_(d)) are extracted by non-linear regressionusing the program Origin 4.1 and the following equation (Gunasekera A,Ebright Y W, Ebright R H (1992) J Biol Chem July 25; 267(21):14713-20)

Ø=[P]/{K _(d) +[P]}

[0175] where Ø denotes the fractional saturation of DNA (i.e. fractionof DNA complexed with the protein). [P] represents the proteinconcentration in the experiment. Ø and [P] were inputs to the non-linearregression; K_(d) was an unconstrained output.

[0176] Various concentrations of Gq1* are incubated with 5′³²-labelled-Htelo, under conditions (150 mM K⁺) that promote andstabilise the G-quadruplex conformation, and the resulting complex isrun on an 8% non-denaturing polyacrylamide gel (see for example FIG.5a). This analysis shows the transition of a low molecular weight bandto a single, higher molecular weight species upon increasing Gq1*concentration.

[0177] The gel mobility shift data was fitted to a quadratic (seeabove—Gunasekera A, Ebright Y W, Ebright R H (1992) J Biol Chem July 25;267(21):14713-20) and equilibrium dissociation constants (K_(d)) areextracted by non-linear regression, to give an observed dissociationconstant (K_(d)) of 34±10 nM (FIG. 5b) which is close to the apparentELISA value (K_(d) ^(E)) of 26 nM. No DNA-binding is observed for GSTprotein alone in the absence of Gq1.

[0178] To elucidate the conformation of the oligonucleotide in theGq1*-Htelo complex, DMS protection experiments are carried out on thecomplexin the form of Dimethyl sulfate protection assay of Htelo andHtelo-Gq1 zinc finger complexes. Htelo is 5′-labelled with ³²P and isdenatured by heating at 95° C. for 10 minutes. Annealing/quadruplexforming reactions are carried out as described above, in 50 mM Tris-HClbuffer with or without 150 mM potassium. DMS protection is carried outas described by Maxam and Gilbert (Maxam, A. M., & Gilbert, W. (1980)Methods Enzymol. 65, 499-560.). 1 μl of dimethylsulfate (DMS) is addedto 20 pmol of annealed Htelo, at 4° C., in 200 μl of appropriate buffer.The mixture is incubated at 20° C. for 5 minutes. Reactions are stoppedby adding ¼ volume of stop buffer containing 1M β-mercaptoethanol and1.5 M sodium acetate, pH 7.0. The reaction products are ethanolprecipitated twice and treated with 100 μl of 1M piperidine at 90° C.for 30 min. The cleaved products are resolved on a 20% denaturingurea-polyacrylamide gel.

[0179] For DMS footprinting of the Htelo-Gq1 zinc finger complex, theprocedure described above was adapted: 2 μl of DMS are added to 0.2 pmolof annealed Htelo, in the absence or presence of 500 nM purified Gq1*(see below), in 200 μl of the appropriate buffer, containing 1 μg/mlcalf thymus DNA. Reactions are carried out for 10 minutes at 20° C.,after which the procedure continues as described above.

[0180] Using 5′ ³²-labelled-Htelo and buffer containing 100 mM K⁺, theconcentration of Gq1* was set at 200 nM which is ˜6-fold higher than theK_(d). These conditions correspond to a near total bandshift (FIG. 5a),representing complete complexation of the DNA.

[0181] In the absence of Gq1*, a cleavage protection pattern isgenerated that is both characterstic of G-quadruplex structure, and thatis dependent on the presence of 100 mM K⁺ (FIG. 6; lanes 1 and 2).However, in the presence of Gq1* and 100 mM K⁺, there is stillsignificant protection of the critical guanines (FIG. 6; lane 3)indicative of G-quadruplex structure. Furthermore, in the absence ofpotassium, the protein does not alter the unfolded state of Htelo (FIG.6; lane 4).

[0182] Thus it is demonstrated that Gq1 binds Htelo in the G-quadruplexconformation, and that this protein molecule according to the inventionrecognises the structure of folded G-quadruplex.

Example 3 Use of Molecules According to the Invention in a TelomeraseAssay

[0183] Telomerase activity may be assayed according to the inventionusing the following method.

[0184] Telomerase template primers are bound to ELISA wells bybiotin-streptavidin linkage as described in Example 2. These primers arenon-G-rich and are not bound by Gq1*.

[0185] Test extracts are added to wells in telomerase extension buffer.

[0186] The test extracts may contain telomerase activity. Such activitywould cause primer extension through the addition of repeats of thesequence [(GGGTTA)n].

[0187] A telomerase extension reaction is carried out in telomeraseextension conditions.

[0188] Telomerase products [(GGGTTA)n] are detected by ELISA asdescribed in Example 2.

[0189] This method provides a convenient and rapid technique for theassay of telomerarse activity, and/or the detection of candidatetelomerase activities.

Sequence Listing

[0190] SEQ. ID. No. 1 GGTTAG GGTTAG GGTTAG GGTTAG GGTTAG SEQ. ID. No. 2TATANNNNNNNGGCGTGTCACAGTCAGCTTCAACGTC SEQ. ID. No. 3TATGTGCGGNNNNNNNTCACAGTCAGTCCACACGTC SEQ. ID. No. 4TATANNNNNNNNNNNNNTCACAGTCAGTCCACACGTC

1. An isolated or purified molecule capable of binding to one or more oftelomeric, G-quadruplex, or G-quartet nucleic acid.
 2. A moleculeaccording to claim 1 wherein said nucleic acid is not in adouble-helical conformation.
 3. A molecule according to claim 1 whereinsaid nucleic acid comprises single-stranded DNA.
 4. A molecule accordingto claim 1 wherein said nucleic acid is comprised in a chromosome end.5. A molecule according to claim 1 wherein said nucleic acid iscomprised in a telomeric structure.
 6. A molecule according to claim 1wherein said nucleic acid is in a non-Watson-Crick base pairedconformation.
 7. A molecule according to claim 1 wherein said nucleicacid comprises Hoogsteen base pairing.
 8. A molecule according to claim1 wherein said molecule is a polypeptide.
 9. A molecule according toclaim 1 wherein said molecule is a polypeptide comprising at least onezinc finger motif.
 10. A molecule according to claim 1 wherein saidmolecule has an affinity for G-quadruplex nucleic acid which isdifferent from its affinity for duplex nucleic acid.
 11. A method forassaying telomerase activity, said method comprising (i) providing asample of nucleic acid substrate for telomerase; (ii) contacting saidnucleic acid sample with a telomerase; (iii) contacting said nucleicacid sample with a molecule according to claim 1; and (iv) monitoringthe binding of said molecule to said telomerase treated nucleic acidsample.
 12. A method according to claim 11 wherein said assay methodcomprises an ELISA assay.
 13. A method according to claim 11 whereinsaid assay method is in micro-well format.
 14. A method for estimatingthe length of telomere(s), said method comprising (i) contacting saidtelomere(s) with a molecule according to claim 1 (ii) monitoring thebinding of said molecule to said telomere sample, and (iii) estimatingthe length of said telomeres from the strength of said binding.
 15. Amethod according to claim 14 wherein said method comprises an ELISAassay.
 16. A method according to claim 14 wherein said method is inmicro-well format.
 17. A method for discriminating between duplex andquadruplex nucleic acid comprising contacting a sample of nucleic acidwith a molecule according to claim 10, and monitoring the binding ofsaid molecule to said nucleic acid.
 18. A method according to claim 17wherein said method comprises an ELISA assay.
 19. A method according toclaim 17 wherein said method is in micro-well format.
 20. A method fordetecting telomeric structures in vivo comprising (i) contacting alabelled molecule according to any preceding claim with a sample, and(ii) monitoring said labelled molecule.
 21. A method according to claim20 wherein said method comprises an ELISA assay.
 22. A method accordingto claim 20 wherein said method is in micro-well format.
 23. A methodfor manipulating telomeric structure(s) in vivo comprising contacting alabelled molecule according to any preceding claim with a telomericstructure, wherein said molecule further comprises an effector domain.